2&#39;,5&#39;-phosphorothioate oligoadenylates and their covalent conjugates with polylysine

ABSTRACT

Optically active compounds of the formula ##STR1## wherein n is 1 or 2 and m is 0, 1, 2 or 3 have antiviral activity. Compounds of the formula wherein at least one of the internucleotide phosphorothioate linkages is of the Sp configuration possess increased antiviral activity and/or metabolic stability.

REFERENCE TO GOVERNMENT GRANT

The invention described herein was made, in part, in the course of worksupported by National Institutes of Health grant POl CA-29545 andNational Science Foundation grant DMB84-15002.

This is a continuation of application Ser. No. 07/499,118, filed on Mar.26, 1990, now abandoned, which is a continuation of application Ser. No.07/112,591, filed on Oct. 22, 1987, now U.S. Pat. No. 4,924,624.

FIELD OF THE INVENTION

The invention relates to synthetic analogues of naturally occurringantiviral 2',5'-oligoadenylates wherein the internucleotidephosphodiester linkages are replaced with optically activephosphorothioate groups. The compounds have increased metabolicstability where the stereoconfiguration around one or more of the chiralphosphorous atoms is the Sp configuration.

BACKGROUND OF THE INVENTION

The full nomenclature of the subject matter of the present inventioninvolves extremely long terms. It is customary for those skilled in theart to abbreviate oligoadenylate analogues and related terms in a mannerwell-known to them. These general and customary abbreviations are setforth herein below and may be utilized in the text of thisspecification.

ABBREVIATIONS

2-5A, 2',5'-oligoadenylate or p₃ A_(n) : Oligomer of adenylic acid with2',5'-phosphodiester linkages and a 5'-terminal triphosphate group.

A₂, A₃ and A₄ : Dimer, trimer and tetramer of adenylic acid with2',5'-phosphodiester linkages.

pA₃, ppA₃ (or p₂ A₃), pppA₃ (or p₃ A₃): 5'-terminal mono-, di- andtriphosphates of A₃.

AMPS: Adenosine 5'-O-phosphorothioate.

SVPD: Snake venom phosphodiesterase.

2'-PDE: 2'-phosphodiesterase

Rp: The R stereoconfiguration about a chiral phosphorous atom in aphosphorothioate internucleotide linkage.

Sp: The S stereoconfiguration about a chiral phosphorous atom in aphosphorothioate internucleotide linkage.

RNase L: 2-5A dependent endoribonuclease.

A_(Rp) A_(Rp) A:(Rp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-adenosine.

A_(Sp) A_(Rp) A:(Sp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-adenosine.

A_(Rp) A_(Sp) A:(Rp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-adenosine.

A_(Sp) A_(Sp) A:(Sp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-adenosine.

pA_(Rp) A_(Rp) A, ppA_(Rp) A_(Rp) A, pppA_(Rp) A_(Rp) A, pA_(Sp) A_(Rp)A, ppA_(Sp) A_(Rp) A, pppA_(Sp) A_(Rp) A, pA_(Rp) A_(Sp) A, ppA_(Rp)A_(Sp) A, pppA_(Rp) A_(Sp) A, pA_(Sp) A_(Sp) A, ppA_(Sp) A_(Sp) A, andpppA_(Sp) A_(Sp) A: 5'-mono-, di- and triphosphates of A_(Rp) A_(Rp) A,A_(Sp) A_(Rp) A, A_(Rp) A_(Sp) A and A_(Sp) A_(Sp) A.

A_(Rp) A_(Rp) A_(Rp) A:(Rp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-adenosine.

A_(Rp) A_(Sp) A_(Rp) A:(Rp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-adenosine.

A_(Rp) A_(Rp) A_(Sp) A:(Rp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')adenosine.

A_(Rp) A_(Sp) A_(Sp) A:(Rp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-adenosine.

A_(Sp) A_(Rp) A_(Rp) A:(Sp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-adenosine.

A_(Sp) A_(Sp) A_(Rp) A:(Sp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-adenosine.

A_(Sp) A_(Rp) A_(Sp) A:(Sp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-adenosine.

A_(Sp) A_(Sp) A_(Sp) A:(Sp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-adenosine.

pA_(Rp) A_(Rp) A_(Rp) A, ppA_(Rp) A_(Rp) A_(Rp) A, pppA_(Rp) A_(Rp)A_(Rp) A, pA_(Rp) A_(Sp) A_(Rp) A, ppA_(Rp) A_(Sp) A_(Rp) A, pppA_(Rp)A_(Sp) A_(Rp) A, pA_(Rp) A_(Rp) A_(Sp) A, ppA_(Rp) A_(Rp) A_(Sp) A,pppA_(Rp) A_(Rp) A_(Sp) A, PA_(Rp) A_(Sp) A_(Sp) A, ppA_(Rp) A_(Sp)A_(Sp) A, pppA_(Rp) A_(Sp) A_(Sp) A, pA_(Sp) A_(Rp) A_(Rp) A, ppA_(Sp)A_(Rp) A_(Rp) A, pppA_(Sp) A_(Rp) A_(Rp) A, pA_(Sp) A_(Sp) A_(Rp) A,ppA_(Sp) A_(Sp) A_(Rp) A, pppA_(Sp) A_(Sp) A_(Rp) A, pA_(Sp) A_(Rp)A_(Sp) A, ppA_(Sp) A_(Rp) A_(Sp) A , pppA_(Sp) A_(Rp) A_(Sp) A, PA_(Sp)A_(Sp) A_(Sp) A, ppA_(Sp) A_(Sp) A_(Sp) A, pppA_(Sp) A_(Sp) A_(Sp) A:5'-mono-, di- and triphosphates of the above tetramers.

(Sp)-ATP-alpha-S: Adenosine 5'O-(sp)-(1-thiotriphosphate).

BACKGROUND OF THE INVENTION

The 2-5A system is widely expected to be involved in the antiviralmechanism of interferon and may also be involved in the regulation ofcell growth and differentiation. 2-5A synthesized from ATP by2',5'-oligoadenylate synthetase [ATP: (2'-5')oligo(A)adenyltransferase(EC 2.7.7.19)] exerts its biological effects by binding to andactivating its only known target enzyme, the unique 2-5A-dependentendoribonuclease RNase L (EC 3.1.27). RNase L cleaves viral and cellularmRNA or rRNA, thereby inhibiting protein synthesis. Hovanessian et al,Eur. J. Biochem. 93:515-526 (1979); Kerr et al, Proc. Natl. Acad. Sci.USA. 75:256-260 (1978). It has been reported that 2-5A protects planttissue from infection by tobacco mosaic virus. Devash et al, Science216:415 (1982). 2-5A, however, is metabolically unstable. It is degradedby a cellular 2'-phosphodiesterase and phosphatases. Knight et al, Meth.Enzymol. 79: 216-227 (1981); Minks et al, Nucleic Acids Res. 6:767-780(1979); Williams et al, Eur. J. Biochem. 92:455-462 (1978).

The literature is replete with structurally-modified 2-5A molecules withmodifications in the adenyl or ribosyl moiety designed to explore thebiological role of the 2-5A synthetase/RNase L system. The primarysource of conformational flexibility in the 2-5A molecule is in thebackbone, similar to 3',5'-linked RNA and DNA. Srinivasan et al, NucleicAcids Res. 13:5707-5716 (1985). However, theoretical and experimentalanalyses have revealed that the conformation of 2',5'-linkeddinucleotides and polynucleotide chains are significantly different from3',5'-linked nucleotides. Id. The ribose-phosphate backbone of 2-5A hasalso been demonstrated to be the major antigenic determinant in themolecule. Johnston et al, Biochemistry 22:3453-3460 (1983).

Few reports have appeared on the synthesis of 2-5A analogues withbackbone modifications. Core analogues containing methylphosphonate andmethylphosphotriester groups have been synthesized. Eppstein et al, J.Biol. Chem. 257:13390-13397 (1982); Jager et al, Nucleic Acids Res. Sym.Ser. No. 9:149-152 (1981). However, complete loss of activity wasobserved with the "uncharged" methylphosphotriester analogues. Eppsteinet al, supra. Substitution of the 2',5'-phosphodiester linkages with 3',5'-linkages has also lead to substantial decrease in biologicalactivity. Lesiak et al, J. Biol. Chem. 258:13082-13088 (1983).Replacement of only one 2',5'-internucleotide linkage has resulted in atleast one order of magnitude loss of activity. Nearly complete loss ofbiological activity was observed when both 2',5'-phosphodiester linkagesin the 2-5A trimer were replaced with 3',5'-bonds.

Haugh et al, Eur. J. Biochem. 132:77-84 (1983), reported that theaffinity of pA₃ to RNase L in mouse L929 cell extracts is approximately1,000 times greater than that of A₃.

Nelson et al, J. Org. Chem. 49:2314-2317 (1984), describe diastereomericpairs of the phosphorothioate analogue of A₃ without resolution ofindividual enantiomers. Eppstein et al, J. Biol. Chem. 261: 5999-6003(1986) report metabolic stabilities and antiviral activity of purportedA_(Rp) A_(Rp) A/A_(Sp) A_(Rp) A and A_(Rp) A_(Sp) A/A_(Sp) A_(Sp) Aracemic mixtures without resolution of individual enantiomers.

Lee and Suhadolnik, Biochemistry 24:551-555 (1985), and Suhadolnik andLee in The 2-5A System: Molecular and Clinical Aspects of theInterferon-Regulator Pathway, Williams, B. R. G. and Silverman, R. H.,Eds. (1985), Alan R. Liss, Inc., New York, p. 115-122, disclose theenzymatic synthesis of the alpha-phosphorothioate 5'-triphosphates ofA_(Rp) A_(Rp) A and A_(Rp) A_(Rp) A_(Rp) A from (Sp)-ATP-alpha-S. Suchcompounds are metabolically unstable. Preparation of the correspondingstereoisomers with Sp internucleotide phosphorothioate linkages was notpossible owing to the stereospecificity of 2-5A synthetase for thesubstrate (Sp)-ATP-alpha-S, which is inverted to yield trimer andtetramer products containing 2',5'-phosphorothioate internucleotidelinkages of the Rp configuration exclusively. Because nucleosidetranferases provide exclusively the inverted configuration whenSp-ATP-alpha-S is the substrate, 2',5'-phosphorothioate oligoadenylatescontaining internucleotide phosphorothioate groups of the Spconfiguration cannot be synthesized enzymatically.

SUMMARY OF THE INVENTION

Compounds of the present invention useful in inhibiting viral infectionsin plants and mammals have increased metabolic stability and/orantiviral activity.

The compounds are optical isomers and water-soluble salts thereof of theformula ##STR2## substantially free of contamination by other opticalisomers of the same formula, wherein m is zero, 1,2 or 3; n is 1 or 2;and at least one of the internucleotide phosphorothioate groups ##STR3##is of the Sp configuration.

The invention also comprises a method of inhibiting viral infection inplants by administering an antiviral effective amount of a compoundaccording to the above formula, or a water-soluble salt thereof, andantiviral compositions containing such compounds with a carrier.

Compounds according to the formula wherein n is 2 may be utilized toform oligoadenylate conjugates with the macromolecular carrierpoly(L-lysine) for intracellular transport. Suchpoly(L-lysine)/2',5'-phosphorothioate oligoadenylate conjugates have theformula ##STR4## wherein q is an integer from about 60 to about 70, andR is randomly R' or ##STR5## From about five to about ten of the Rgroups comprise R'. R' has the following formula wherein m is 0,1,2 or3: ##STR6##

Preferably, at least one of the phosphorothioate groups ##STR7## of thepoly(L-lysine)/2',5'-phosphorothioate oligoadenylate conjugates is ofthe Sp configuration.

DETAILED DESCRIPTION OF THE INVENTION

The activation of the unique 2-5A-dependent endoribonuclease, RNase L,by 2',5'-oligoadenylates, and its hydrolysis of rRNA, tRNA and cellularand viral mRNA, is important in the inhibition of viral replication andregulation of cell growth. RNase L is known to be the substrate targetfor 2-5A. It is one of the chief functional enzymes of theinterferon-induced biological cascade. While modified analogues of 2-5Ahave been reported, they are not metabolically stable, or fail toactivate RNase L. The introduction of the phosphorothioate group in the2',5'-internucleotide linkages of 2-5A, induces physical, chemical andbiochemical modifications in the 2-5A molecule including (i) Rp/Spchirality, (ii) lowered pKa, (iii) altered metal ion chelation, (iv)charge modulation, (v) increased bond length, (vi) altered degree ofhydration and (vii) increased metabolic stabilities.

The metabolic stability of the 2',5'-phosphorothioate oligoadenylates isgreater than authentic 2-5A. This metabolic stability is greatlyenhanced where at least one of the internucleotide phosphorothioate2',5'-linkages is of the Sp configuration. While racemic mixtures of thetrimer cores have been reported, Nelson et al, J. Org. Chem.49:2314-2317 (1984) and Eppstein et al, J. Biol. Chem 261:5999-6003(1986), efforts to resolve the compounds have failed. The level ofantiviral activity of the trimer core racemates reported by Eppstein etal is such that the dosages required for treatment would beprohibitively toxic. At least one of the purported racemates of Eppsteinet al, the A_(Rp) A_(Sp) A/A_(Sp) A_(Sp) A racemate, is of little valuesince, as we have found, the A_(Sp) A_(Sp) A stereoisomer selectivelyinactivates RNase L, thereby preventing A_(Rp) A_(Sp) A from exertingits antiviral effect through activation of RNase L. This is mostundesirable since, as we have found, A_(Rp) A_(Sp) A is the mostattractive of the four trimer core stereoisomers, since it bothactivates RNase L and is metabolicly stable.

We have succeeded in preparing the fully resolved 2',5'-phosphorothioateadenylate trimer cores, thus making possible the practical use of theimportant A_(Rp) A_(Sp) A stereoisomer. Our method of stereo-specificchemical synthesis also makes possible the preparation of the eightseparate stereoisomers of the 2',5'-phosphorothioate tetramer.Preparation of the tetramer molecules enables conjugation with thecarrier (poly)L-lysine, shown to be an effective vector for introducing2',5'-oligoadenylates and analogues into intact cells. Poly(L-lysine)conjugation to trimer molecules is not feasible, owing to thedestruction of the 2'-terminal ribosyl moiety and subsequentinactivation of the molecule. Conjugation to poly(L-lysine) permitsefficient intracellular transport of the 2',5'-phosphorothioateoligoadenylates while preserving intact within the conjugate the trimermoiety believed necessary for good biological activity.

Correlation of biological properties with absolute configuration hasonly been possible with the preparation of the fully resolved2',5'-phosphorothioate adenylate trimer cores described herein. However,the trimer core compounds have been found to bind and/or activate RNaseL only modestly. We have found that the RNase L activation by the2',5'-phosphorothioate core molecules is significantly enhanced by5'-phosphorylation.

RNase L activation by authentic 2-5A requires the triphosphate form ofthe trimer. The 5'-monophosphate form of 2-5A is a potent inhibitor ofthe RNase L-activating activity of the triphosphate. Miyamoto et al., J.Biol. Chem 258: 15232-15237 (1983); Black et al., FEBS Let. 191:154-158(1985); Torrence et al., Proc. Natl. Acad. Sci. USA 78:5993-5997 (1981).We have surprisingly found that the cores and monophosphates of thepresent phosphorothioate analogues of 2-5A, unlike authentic 2-5A,activate RNase L.

The phosphorothioate trimer cores A_(Rp) A_(Rp) A, A_(Sp) A_(Rp) A,A_(Rp) A_(Sp) A, and A_(Sp) A_(Sp) A, are chemically synthesized andseparated by preparative thin layer chromatography on silica gel. Thefour trimer cores are prepared from6-N-benzoyl-3'-O-tert-butyldimethylsilyl-5'-O-monomethoxytrityladenosine-2-O-(p-nitrophenylethyl)-octahydroazonino-phosphoramiditeby stereospecific synthesis, which relies on separation of fullyresolved protected intermediates followed by removal of all blockinggroups to yield the fully-resolved 2',5'-phosphorothioate trimeradenylate cores.

The trimer cores are prepared according to the following reaction schemewherein "BZ" denotes the benzoyl radical "Si" denotes thetert-butyldimethylsilyl radical and "MMTr" represents themonomethoxytrityl radical. While not part of the invention, thepreparation of the dimer core enantiomers A_(Rp) A (6A) and A_(Sp) A(6B) is included for completeness. ##STR8##

The blocking groups are removed from the fully-protected intermediates7A, 7B, 8A, and 8B to yield the corresponding fully resolved trimercores A_(Rp) A_(Rp) A (9A), A_(Sp) A_(Rp) A (9B), A_(Rp) A_(Sp) A (10A)and A_(Sp) A_(Sp) A (10B): ##STR9##

The compounds of the invention are advantageously prepared as solublesalts of sodium, ammonium or potassium. The preparative scheme beginswith6-N-benzoyl-3'-0-tert-butyldimethylsilyl-5'-0-monomethoxy-trityladenosine(compound 1E), which is advantageously prepared from adenosine accordingto the procedure of Flockerzi et al, Liebigs Ann. Chem., 1568-1585(1981).

Preparation of the compounds of the present invention is illustrated inmore detail by reference to the following non-limiting examples.3-Nitro-1,2,4-triazole;chlorooctahydroazonino-p-nitrophenylethoxyphosphate;2,5-dichlorophenylphosphoro-dichloridate; and p-nitrophenylethanol usedin the examples may be prepared advantageously from publishedprocedures: Chattopadhyaya et. al. Nucleic Acids Res. 8:2039-2053(1980); Schwarz et al., Tetrahedron Lett. 5513-5516 (1984); Uhlmann etal., Helv. Chim. Acta 64:1688-1703 (1981). These compounds are alsoavailable commercially in the United States.2,5-Dichlorophenyl-phosphorodichloridate may be obtained from FlukaChemical Corp., 980 S. Second St., Ronkonkoma, N.Y. 11779, ("Catalog 15:Chemika-Biochemika" 1986/1987, no. 36212). 3-Nitro-1,2,4-triazole isavailable from Aldrich Chemical Co., P.O. Box 355, Milwaukee, Wisc.53201 (1986-1987 cat. no. 24,179.2). P-Nitrophenylethanol is availablefrom Fluka Chemical Corp. (cat. no. 73,610).Chloro-octahydroazonino-p-nitrophenylethoxyphosphate may be preparedaccording to Example 1A, below.

Pyridine and triethylamine used in the examples were purified bydistillation over KOH, tosyl chloride and calcium hydride.Dichloromethane was distilled over calcium chloride and then passedthrough basic alumina. Pure acetonitrile was obtained by distillationover calcium hydride.

Purification of the protected nucleotides was achieved by preparativecolumn chromatography on silica gel 60 (0.063-0.2 mesh, Merck) and bypreparative thick layer chromatography on silica gel 60 PF₂₅₄ (Merck).Thin layer chromatography ("TLC") was carried out on precoated thinlayer sheets F 1500 LS 254 and cellulose thin layer sheets F 1440 fromSchleicher & Scheull.

The starting material,6-N-Benzoyl-3'-0-tert-butyldimethylsilyl-5'-0-(4-monomethoxytrityl)-adenosine(Compound 1E) and the reagent6-N-benzoyl-2',3'-bis-0-(tert-butyldimethylsilyl)-adenosine (Compound1G), are prepared according to Example 1.

EXAMPLE 1

a N⁶,N⁶,2',3',5'-O-Pentabenzoyladenosine: (1A) To a suspension of 5.34 g(20 mmole) adenosine (Sigma, dried at 80° C./10⁻³ Torr for 24 h) in 100ml dry pyridine, 33.74 g (240 mmole) benzoyl chloride was addeddropwise. After 20 h stirring at room temperature ("r.t.") the mixturewas treated with 16 ml dry MeOH and then extracted with CHCl₃ (3×250ml). The organic phase was washed with water (3×250 ml), dried over Na₂SO₄, and evaporated to dryness. Final coevaporation was performed withtoluene. The residue was dissolved in CHCl₃ /MeOH 2/1 by heating, andafter cooling to r.t., petrolether (diethylether) was added until thesolution became turbid. After standing for 12 h at 0° C. 12.18 g wasobtained, and from the mother liquor, 2.66 g of the product wereisolated as colorless needles of m.p. 183°-184° C., yield 14.84 g (94%).

b. 6-N-Benzoyl adenosine (1B): A solution of 7.88 g (10 mmole ) of thepentabenzoyl adenosine (1A) in 150 ml dry pyridine and 50 ml dry MeOHwas treated with 50 ml 1M sodium methylate solution. After 15 min thesolution was poured onto an ice cold solution of a 110 ml DOWEX ionexchanger 50×4 (pyridinium form) in ca 20 ml water. After 5 h stirringthe pH was 5.5-6.0. After filtering from the ion exchanger, the residuewas washed with boiling MeOH/water (3/1). The filtrate was evaporated todryness and crystallized from MeOH/water 2/1 to give 3.25 g (83% ) ofthe product as colorless needles, m.p. 151°-153° C.

c. 6-N-Benzoyl-5'-O-(4-methoxytrityl) adenosine (1C): 17.9 g (46 mmole)6-N-benzoyl adenosine.H₂ O (1B) was evaporated with dry pyridine (3×100ml) and finally dissolved in 150 ml dry pyridine and 21.31 g (69 mmole)p-monomethoxytrityl chloride. The reaction solution was stirred at 50°C. for 14 h and dry MeOH (50 ml) was added and allowed to come to r.t.The product was extracted with CHCl₃ (3×400 ml) and washed with water(3×400 ml). The organic phase was dried over Na₂ SO₄ and evaporated todryness. Final coevaporation was performed with toluene. Purificationwas performed using a silica gel column (16×2.5 cm, Merck) and elutedwith 3 liter EtOAc/MeOH 7/3 to give 25.6 g (87%) of an amorphous powder.Crystallization was accomplished with acetone/water to yield theproduct, m.p. 120°-125° C.

d. 6-N-Benzoyl-2'-O-(tert-butyldimethylsilyl)-5'-O-(4-methoxytrityl)adenosine (1D);

e. 6-N-Benzoyl-3'-O-(tert-butyldimethylsilyl)-5'-O-(4-methoxytrityl)adenosine (1E);

6-N-benzoyl-2',3'-O-bis(tert-butyldimethylsilyl)-5'-O-(4-methoxytrityl)adenosine (1F):

To a solution of 4.05 g (26.9 mmole ) tert-butyldimethylsilylchloride("TBDMS-Cl") and 3.66 g (53.8 mmole) imidazole in 100 ml dry pyridine,were added 14.42 g (22.4 mmole) of compound 1C which was previouslycoevaporated with dry pyridine. After 15 h stirring at r.t., 5 ml dryMeOH was added and the mixture was evaporated to 1/3 volume. The crudeproduct was extracted with CHCl₃ (3×250 ml) and washed with water. Uponevaporation, the crude product was first purified by a silicagel column(15×3 cm) with CH₂ Cl₂ /EtOAc (9/1), and subsequently purified usingmedium pressure chromatography (silicagel column, GSF-type C (N=9000,V_(D) =28 ml) at 8-10 bar pressure with the following mixtures of CH₂Cl₂ /petrolether/EtOAc/EtOH: 100:100:10:0.5 (2 liters), first;100/100/10/1 (3 liters), second; and 100/100/31/2 (0.5 liters), third.The title compounds were isolated. The retention time of the peak maximafor each compound was as follows: 25 min. for compound 1F (yield 1.84 g,9%); 60 min. for compound 1D (yield 6.62 g, 39%); 105 min. for compound1E (yield 8.32 g, 49%).

e 6-N-Benzoyl-2',3'-bis-O-(tert-butyldimethylsilyl) adenosine (1G): 1.74g (2 mmole) of compound 1D was stirred with 20 ml 80% acetic acid at 22°C. After 20 h the cleavage of the monomethoxytrityl group was complete.The reaction mixture was extracted with CHCl₃ (3×200 ml) and washed with200 ml 1M phosphate buffer (pH 7). The organic phase was dried over Na₂SO₄ and evaporated to dryness. Purification was accomplished using asilica gel column (2×10 cm) and eluted with CH₂ Cl₂ /MeOH (96/4). Thelight yellow product was dissolved in 5 ml CHCl₃ and treated with Et₂ Ountil turbid. 0.982 g of the pure product crystallized out. The pureproduct crystallized out again from the mother liquor, 0.11 g, m.p. 189°C. The total yield was 1.092 g (91%).

EXAMPLE 1A Chloro-octahydroazonino-p-nitrophenylethoxyphosphate

a. P-nitrophenylphosphoric acid dichloride. To phosphorus trichloride(Fluka, N.Y., #79690) (28 ml, 0.317 moles) 80 ml anhydrous ether areadded. The mixture is cooled to -30° C. P-nitrophenylethanol (8.35 g, 50mmoles) is added, followed by stirring for 1.5 hr. Ether and excess PCl₃is removed under vacuum to yield p-nitrophenylphosphoric acid dichloride(yield 80%).

b. Octahydroazonin. Caprylolactam (Fluka, N.Y., #21631) (25 g, 117mmoles) is combined with lithium aluminum hydride (10.5 g) in ether andreduced with stirring for 5 hr. The reaction mixture is filtered andevaporated with ether. The product is octahydroazonin (90% yield).

c. 1-Trimethylsilyl octahydroazonin. The silylamine of octahydroazoninis prepared by combining octahydroazonin (12.7 g, 0.1 moles) andtrimethylsilane (0.12 moles) and 0.5 moles hexamethyldisilazane+150 mgof ammonium sulfate. The mixture is refluxed for 90 hr and distilledunder vacuum to yield 16.5 g of 1-trimethylsilyl octahydroazonin (82% ).

d. Chloro-octahydroazonino-p-nitrophenylethoxyphosphate.P-nitrophenylphosphoric acid dichloride (26.8 g, 100 mmoles) and1-trimethylsilyl octahydroazonin (19.9 g, 100 mmoles) are combined undernitrogen at 0° C. The mixture is warmed to room temperature and stirred2-3 h. Trimethylsilyl dichloride is removed under vacuum. The product inthe residue is chloro-octahydroazonino-p-nitrophenylethoxyphosphate(33.9 g, 94% yield).

EXAMPLE 2

6-N-Benzoyl-3'-0-tert-butyldimethylsilyl-5'-0-(4-ethoxytrityl)-adenosine-2'-0-(p-nitrophenylethyl)-octahydroazonino-phosphoramidite(2). Compound 1E (0.758 g, 1.0 mmole) and diisopropylethylamine (0.52 g,4 mmole) were dissolved in dichloromethane (5 ml) andchlorooctahydroazonino-p-nitrophenylethoxyphosphane (0.80 g, 2.22 mmole)was added dropwise. After stirring for 2 h at r.t., TLC analysisindicated complete reaction. The reaction mixture was transferred to aseparatory funnel using saturated aqueous NaHCO₃ (50 ml) and the productwas isolated by extraction with ethylacetate (2×50 ml). The organiclayer was washed with saturated NaCl, dried (Na₂ SO₄), and evaporated todryness. The residue was dissolved in ethylacetate-triethylamine (95:5v/v), chromatographed on a silica gel column (10×2 cm) previouslycalibrated with ethylacetate-triethylamine (9/1) and eluted withethyl-acetate-triethylamine (95:5 v/v). The product fractions werecollected, evaporated to dryness, finally coevaporated withdichloromethane and dried in vacuo at 40° C. to give compound 2 (1.05 g,97%) [Anal. calcd. for C₅₉ H₇₀ N₇ O₉ PSi . 1 H₂ O: C, 64.52; H, 6.60; N,8.92. Found: C, 63.93; H, 6.85; N, 8.62].

EXAMPLE 3

6-N-Benzoyl-3'-0-tert-butyldimethylsilyl-5'-0-monoethoxytrityl-P-thioadenylyl-2'-[0^(P)-(p-nitrophenylethyl)-5']-6-N-benzoyl-2',3'-di-0-tert-butyldimethylsilyladenosine(4A+4B). The phosphoramidite 2 (1.12 g, 1.0 mmole) and6-N-benzoyl-2',3'-di-tert-butyldimethylsilyladenosine (3) (0.478 g, 0.7mmole) were dried overnight in a drying pistol at 40° C. in vacuo. Thedried residue was then dissolved in dry acetonitrile (6 ml), and3-nitro-1,2,4-triazole (0.285 g, 2.5 mmole) was added and stirred atr.t. for 3 h. Pyridine (6 ml) and sulfur (0.5 g) were added and afterstirring at r.t. for 20 h, the reaction mixture was extracted withchloroform (300 ml). The organic phase was washed with saturatedNaCl-solution (2×200 ml), dried (Na₂ SO₄), and evaporated to dryness.Final evaporation was performed with toluene to remove pyridine. Theresidue was dissolved in chloroform and chromatographed on a silica gelcolumn (15×2.5 cm) with 1 liter of chloroform to give a product fractioncontaining both Rp and Sp isomers. The separation of thediastereoisomers was carried out on preparative silica gel plates, usingdichloromethane/ethylacetate/n-hexane (1:1:1 v/v). The plates weredeveloped thrice. The higher Rf isomer (0.47 g; 42%, TLC indichloromethane/ethylacetate/n-hexane, 1:1:1, 0.54) and the lower Rfisomer (0.31 g; 28%, TLC in dichloromethane/ethylacetate/n-hexane, 0.46)were obtained as colorless amorphous powders after drying at 40° C. invacuo. The higher Rf isomer was compound 4A. [Anal. calcd. for C₈₀ H₉₈N₁₀ O₁₄ PSi₃ . 1 H₂ O: C, 59.89; H, 6.32; N, 9.61. Found: C, 59.89; H,6.32; N, 9.21]. ³¹ P-NMR (400 MHz, CDCl₃, 85% H₃ PO₄, 69.841 ppm). Thelower Rf isomer was compound 4B. [Anal. calcd. for C₈₀ H₉₈ N₁₀ O₁₄ PSi₃S . 1 H₂ O: C, 59.89; H, 6.28; N, 9.61. Found: C, 59.91; H, 4.61; N,9.28]. ³¹ P-NMR (400 MHz, CDCl₃, 85% H₃ PO₄, 69.223 ppm).

EXAMPLE 4

6-N-Benzoyl-3'-O-tert-butyldimethylsilyl-P-thioadenylyl-2'-[0^(P)-(p-nitrophenylethyl)-5']-6-N-benzoyl-2',3'-di-0-tert-butyldimethylsilyladenosine (5A+5B).0.258 g, (0.164 mmole) of the pure isomers 4A and 4B, were detritylatedseparately by treatment of each with 2% p-toluenesulfonic acid indichloromethane/methanol (4/1) (3.2 ml) at r.t. for 40 min. The reactionmixture was diluted with chloroform (50 ml), washed with phosphatebuffer, pH 7 (2×20 ml), and evaporated to a foam. The residue waspurified by silica gel column chromatography (10×2.5 cm). Compounds 5Aand 5B were separated on the column by using chloroform andchloroform/methanol (100:0.5 till 100:1). The product fractions werecollected and, after evaporation, dried in vacuo at 40° C. to give inthe case of 5A, 0.198 g (92%), and in the case of 5B, 0.192 g (89%). 5Ahas an Rf of 0.27 in dichloromethane/ethylacetate/n-hexane (1:1:1).[Anal. calcd. for C₆₀ H₈₂ N₁₁ O₁₃ PSi₃ S . 1 H₂ O: C, 54.15; H, 6.36; N,11.57. Found: C, 53.78; H, 6.44; N, 11.72]. 5B has an Rf of 0.30 in thesame system. [Anal. calcd. for C₆₀ H₈₂ N₁₁ O₁₃ PSi₃ S: C, 54.90; H,6.29; N, 11.73. Found: C, 54.90; H, 6.20; N, 11.45].

EXAMPLE 5

P-Thioadenylyl-(2'-5')-adenosine (6A+6B). The fully protected dimers 5Aand 5B, respectively, were deprotected separately by the followingprocedure. Each protected dimer (39 mg, 0.03 mmole ) was treated with0.5M 1,8-diazabicyclo[5.4.O]undec-7-ene(1,5,5)(Fluka, cat. no. 33842,"DBU") in pyridine (9 ml), and after stirring at r.t. for 2 h wasneutralized with 1M acetic acid (4.5 ml) and finally evaporated. Theresidue was taken up in 1M tetrabutylammonium fluoride ("Bu₄ NF") intetrahydrofuran ("THF") and after 24 h again evaporated to dryness.Deacetylation was achieved by treatment with conc. ammonia (20 ml) for48 h followed by evaporation of the mixture. The residue was thendissolved in water (50 ml) and washed with chloroform (2×20 ml). Thewater phase was applied to a DEAE Sephadex A-25 column (60×1 cm) forelution with a linear gradient of a buffer of 0.001-0.25M Et₃ NH⁺ HCO₃⁻, pH 7.5. The product was eluted at a concentration of 0.08-0.1M.Evaporation to dryness followed by coevaporation with water (10×10 ml)and final purification by paper chromatography with i-PrOH/conc.ammonia/water (7:1:2 v/v) yielded in the case of 6A 630 O.D. units(87.5%) and in the case of 6B 648 O.D. units (90%). The Rf of 6A oncellulose sheets, using the above system, was 0.29. The Rf of 6B was0.30.

EXAMPLE 6

6-N-Benzoyl-3'-0-tert-butyldimethylsilyl-5'-0-monomethoxytrityl-P-thioadenylyl-2'-[⁰P-(p-nitrophenylethyl)-5']-N-6-benzoyl-3'-0-tert-butyldimethylsilyl-P-thioadenylyl-2'-[0^(P)-(p-nitrophenylethyl)-5']-6-N-benzoyl-2',3'-bis-0-tert-butyldimethylsilyladenosine(7A, 7B and 8A, 8B). The phosphitamide 2 (0.449 g; 0.41 mmole) wascondensed with the 5'-hydroxy dimer 5A and 5B (0. 0262 g; 0.2 mmole)separately in the presence of 3-nitro-1,2,4-triazole (0.114 g; 1.0mmole) in dry acetonitrile (3.2 ml). After stirring at r.t. for 3 h,sulfur (0.2 g; 6.25 mmole) in pyridine (0.4 ml) was added for oxidation.After stirring at r.t. for another 24 h, the product was extracted withdichloromethane (50 ml), the organic phase was washed with saturatedNaCl solution (2×20 ml), dried (Na₂ SO₄), and then evaporated todryness. Final coevaporation was performed with toluene to removepyridine. The crude product was chromatographed on a silica gel column(15×2 cm) and eluted with chloroform/methanol (100:2) to give, oncondensing with compound 5A, the isomer mixture 7A+7B. The isomermixture 8A+8B, was obtained upon condensing the phosphitamide 2 withcompound 5B in the same manner. The diastereomeric separation of eachisomer mixture was accomplished by using preparative silica gel plates(20×20×0.2 cm) to which about 50 mg of isomer mixture per plate wasapplied for optimal separation. The plates were developed indichloromethane/ethylacetate/n-hexane (1:1:0.5 v/v) three times. Theappropriate bands were cut out and eluted with chloroform/methanol(4:1). The higher Rf isomer synthesized from 5A has a Rf of 0.58 indichloromethane/ethylacetate/n-hexane (1:1:1) and yielded 48% (0.218 g)of 7A. [Anal. calcd. for C₁₁₁ H₁₃₅ N₁₇ O₂₂ P₂ Si₄ S₂ . 1 H₂ O: C, 57.56;H, 5.96; N, 10.28. Found: C, 57.38; H, 5.99; N, 10.11]. The lower Rfisomer has a Rf of 0.48 in the above-mentioned system, and yielded 34%(0.157 g) of 7B. [Anal. calcd. for C₁₁₁ H₁₃₅ N₁₇ O₂₂ P₂ Si₄ S₂ . 1 H₂ O:C, 57.56; H, 5.96; N, 10.28. Found: C, 57.40; H, 5.97; N, 10.19].

The isomeric mixture derived from the 5'-hydroxy dimer 5B was separatedin the same manner and yielded the higher Rf isomer 8A in 41% (0.186 g)with an Rf of 0.53 in the above solvent system. [Anal. calcd. for C₁₁₁H₁₃₅ N₁₇ O₂₂ P₂ Si₄ S₂ : C, 58.02; H, 5.92; N, 10.36. Found: C, 58.00;H, 5.84; N, 10.66]. The lower Rf isomer 8B showed a Rf value of 0.45 andyielded 35% (0.161 g). [Anal. calcd. for C₁₁₁ H₁₃₅ N₁₇ O₂₂ P₂ Si₄ S₂ . 1H₂ O: C, 57.56; H, 5.96; N, 10.28. Found: C, 57.30; H, 5.78; N, 10.03].

EXAMPLE 7

(Rp)-P-Thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-adenosine (9A).A solution of 0.116 g (0.05 mmole of the fully protected trimer 7A wasdetritylated with 2% p-toluenesulfonic acid in 1.5 mldichloromethane/methanol (4:1) for 90 min. The mixture was dissolved inCHCl₃, washed with phosphate buffer (2×15 ml), dried (Na₂ SO₄), andevaporated to dryness. The residue was chromatographed on silica gelplates (20×20×0.2 cm), developed withdichloromethane/ethylacetate/n-hexane (5:5:3 v/v). The product band(R_(f) 0.35) was cut out, eluted with chloroform/methanol (7:5) and gaveon evaporation to a colorless foam a yield of 70-83%. 38.5 mg (18.9micromole) of the product was then stirred with 0.5M DBU in pyridine(7.5 ml) for 20 h, neutralized with 1M acetic acid (3.75 ml) and finallyevaporated. The evaporated product was desilylated through treatmentwith 1M Bu₄ NF in THF (6 ml) for 24 h. The mixture was concentrated invacuo. The residue was dissolved in conc. ammonia (25 ml) and stirred atr.t. for 48 h. After evaporating the solution, the residue was taken upin water (20 ml) and washed with chloroform (2×10 ml). The aqueous phasewas put on a DEAE Sephadex A-25 column (60×1 cm) and the product waseluted with a linear gradient of Et₃ NH⁺ HCO₃ -buffer. The productfraction was collected, evaporated and further purified by paperchromatography using i-PrOH/conc. ammonia/water (6:1:3) to give thetitle compound in 75-80% yield.

EXAMPLE 8

(Sp)-P-Thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-adenosine (9B).A solution of 0.116 g (0.05 mmole) of the fully protected trimer 7B wassubjected to the procedure of Example 7. The title compound was obtainedin 75-80% yield.

EXAMPLE 9

(Rp)-P-Thioadenylyl-(2'-5')-(Sp)-P-thiodenylyl-(2',5')-adenosine (10A).A solution of 0.116 g (0.05 mmole) of the fully protected trimer 8A wassubjected to the procedure of Example 7. The title compound was obtainedin 75-80% yield.

EXAMPLE 10

(Sp)-P-Thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-adenosine (10B).A solution of 0.116 g (0.05 mmole) of the fully protected trimer 8B wassubjected to the procedure of Example 7. The title compound was obtainedin 75-80% yield.

The UV-absorption spectra in methyl alcohol and ¹ H-NMR spectra of theabove-prepared protected monomer, dimer and trimer cores are set forthin Tables 1 and 2, respectively.

                  TABLE 1                                                         ______________________________________                                        UV-Absorption Spectra of Protected                                            Monomer, Dimer and Trimer Cores in MeOH                                       Compound     λ.sub.max                                                                     (nm)            lgε                               ______________________________________                                        2            230    277        4.47 4.50                                      4A           231    277        4.66 4.70                                      4B           231    277        4.66 4.70                                      5A                  278             4.70                                      5B                  278             4.70                                      7A           230    278        4.78 4.90                                      7B           230    278        4.78 4.89                                      8A           230    278        4.79 4.90                                      8B           230    278        4.78 4.90                                      ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    .sup.1 H-NMR Spectra of Protected                                             Monomer, Dimer and Trimer Cores.sup.1                                         Compound                                                                            1'-H        2-H      8-H      Solvent                                   __________________________________________________________________________    2     6.11                                                                              6.13    8.70     8.23     CDCl.sub.3                                4A    6.30 d                                                                            5.87 d  8.68;                                                                            8.60  8.19;                                                                            8.17  CDCl.sub.3                                4B    6.29 d                                                                            5.94 d  8.72;                                                                            8.62  8.26;                                                                            8.18  CDCl.sub.3                                5A    6.06 d                                                                            5.94 d  8.82;                                                                            8.75  8.25;                                                                            8.08  CDCl.sub.3                                5B    6.13 d                                                                            5.90 d  8.74;                                                                            8.73  8.26;                                                                            8.24  CDCl.sub.3                                7A    6.23 d                                                                            6.08 d                                                                            5.84 d                                                                            8.69;                                                                            8.58;                                                                            8.55;                                                                            8.20;                                                                            8.11;                                                                            8.01                                                                             CDCl.sub.3                                7B    6.22 d                                                                            6.17 d                                                                            5.85 d                                                                            8.67;                                                                            8.60;                                                                            8.57;                                                                            8.23;                                                                            8.10;                                                                            8.00                                                                             CDCl.sub.3                                8A    6.27 d                                                                            6.13 d                                                                            5.93 d                                                                            8.71;                                                                            8.61;                                                                            8.60;                                                                            8.21;                                                                            8.13;                                                                            8.00                                                                             CDCl.sub.3                                8B    6.27 d                                                                            6.22 d                                                                            5.90 d                                                                            8.71;                                                                            8.64;                                                                            8.61;                                                                            8.27;                                                                            8.22;                                                                            8.19                                                                             CDCl.sub.3                                __________________________________________________________________________     .sup.1 δ values in ppm; Standard TMS; characteristic signals       

Assignment of Absolute Configuration of 2',5'-Phosphorothioate AdenylateTrimer Cores

Determination of the absolute configurations of the trimer cores wasaccomplished by ³¹ P-NMR fast bombardment mass spectrometry andenzymatic digestion.

It is known that the enzyme SVPD preferentially cleaves Rp-3',5'- or2',5'-phosphorothioate linkages from the 2'/3'-terminus Nelson et al, J.Org. Chem. 49:2314-2317 (1984); Eppstein et al, J. Biol. Chem.261:5999-6003 (1986); Lee et al, Biochemistry 24:551-555 (1985). SVPDhydrolysis of the chemically synthesized dimer core A_(Rp) A yieldedadenosine and AMPS in a molar ratio of 1:1, respectively; the half-lifewas 3 hours (Table 3). The A_(Sp) A dimer core was not a substrate forSVPD under these conditions. Trimer core 9A has the RpRp internucleotidelinkage configuration as determined by hydrolysis by SVPD to yield AMPSplus A_(Rp) A in a molar ratio of 1:1, respectively. Similarly, SVPDhydrolysis of trimer core 9B yielded A_(Sp) A and AMPS, thus identifyingtrimer core 9B as having the SpRp internucleotide linkage configuration(Table 3). Trimer cores 10A and 10B were not substrates for SVPD (Table3), revealing the presence of the Sp configuration in theinternucleotide linkage adjacent to the 2'/3'-termini.

                                      TABLE 3                                     __________________________________________________________________________    Analytical Data, Dimer and Trimer 2,5-Phosphorothioate Adenylate Cores        2,5-Phosphorothioate                                                                                                serum phospho-                                                                         Stereo-                                           SVPD     L cell extract                                                                          diestereases                                                                           config-                               .sup.31 P-NMR                                                                         R.sub.T                                                                           dimer core                                                                          half                                                                             dimer core                                                                          half                                                                              dimer core                                                                          half                                                                             uration                               (PPM.sup.a)                                                                           (min.sup.d)                                                                       isolated                                                                            life                                                                             isolated                                                                            life                                                                              isolated                                                                            life                                                                             assigned                       __________________________________________________________________________    Dimer Cores                                                                   6A     57.63   19.5                                                                              --    3 h                                                                              not cleaved                                                                             not cleaved                                                                            Rp                             6B     56.13   24.2                                                                              not cleaved                                                                            not cleaved                                                                             not cleaved                                                                            Sp                             Trimer Cores                                                                  9A     57.45,                                                                             57.71                                                                            30.5                                                                              Rp    1 h                                                                              Rp    18 h                                                                              Rp    8 h                                                                              RpRp.sup.b                     9B     57.55,                                                                             56.62                                                                            33.0                                                                              Sp    8 h                                                                              Sp    15 h                                                                              not cleaved                                                                            SpRp.sup.c                     10A    56.34,                                                                             57.54                                                                            35.2                                                                              not cleaved                                                                            Rp    20 days                                                                           not cleaved                                                                             RpSp.sup.c                    10B    56.50,                                                                             56.26                                                                            39.5                                                                              not cleaved                                                                            not cleaved                                                                             not cleaved                                                                            SpSp.sup.b                     A.sub.3 core                                                                              14.5                                                                             --  5 min -- 10 min                                                                              --  10 min                                  A.sub.2 core       ND       --    10 min                                                                            ND                                      __________________________________________________________________________     .sup.a decoupled spectra                                                      .sup.b assignment confirmed by coupled and decoupled .sup.31 PNMR             .sup.c assignment confirmed by coupled .sup.31 PNMR and enzymatic             hydrolyses                                                                    .sup.d HPLC retention times                                              

The four 2,5-phosphorothioate adenylate trimer cores were furthercharacterized by hydrolysis with the enzyme 2'-phosphodiesterase("2'-PDE"), an exoribonuclease found in L cell extract. The enzymecleaves from the 2'/3'-terminus. Whereas authentic A₂ and A₃ cores wherehydrolyzed to adenosine and AMP with a half-life of 10 min, the dimercores A_(Rp) A and A_(Sp) A were not substrates for 2'-PDE (Table 3).The fact that the 2,5-phosphorothioate dimer cores were not substratesfor 2'-PDE (unlike authentic 2-5A) greatly assisted in the assignment ofthe stereoconfigurations of the 2',5'-phosphorothioate adenylate trimercores. Trimer core 9A was a substrate for 2'-PDE; the products ofhydrolysis were A_(Rp) A and AMPS. Trimer core 9B was a substrate forSVPD, yielding A_(Sp) A and AMPS; trimer core 10A was a substrate,yielding A_(Rp) A and AMPS; trimer 10B was not a substrate for 2'-PDE.

³¹ P-NMR spectroscopy has revealed that Sp stereoisomers ofphosphorothioates resonate to higher field than Rp diastereomers.Further, Sp diastereomers have a longer retention time on reverse phaseHPLC than Rp diastereomers. The A_(Sp) A dimer core resonates upfieldfrom the A_(Rp) A (Table 3). Similarly, two singlets observed for A_(Sp)A_(Sp) A resonate upfield from the two singlets observed for A_(Rp)A_(Rp) A (Table 3). Assignment of the absolute configurations of trimercores 9A (RpRp) and 10B (SpSp) was based on the two singlets whichresonate at the same frequency as the singlets observed for the A_(Rp) Aand A_(Sp) A dimer cores. Assignment of configurations for trimer cores9B and 10A was made in combination with the enzyme degradations and HPLCanalyses (Table 3). The ³¹ P-NMR spectra revealed that the δppm betweenthe two singlets for the A_(Sp) A_(Rp) A trimer core is 1.2, whereas thetwo singlets for the A_(Rp) A_(Sp) A have a δppm of 0.8 (assignment is 5' to 2'/3' terminus).

The metabolic stability of the 2,5'-phosphorothioate dimer and trimercores is markedly greater than authentic 2-5A. The rate of hydrolysis ofthe trimer cores by the 3'-exonuclease SVPD is, in order of decreasingstability: A_(Sp) A_(Rp) A>A_(Rp) A_(Rp) A>>>A₃. The trimer cores A_(Rp)A_(Sp) A and A_(Sp) A_(Sp) A are not substrates of SVPD (Table 3). The3'→'5'direction of stepwise cleavage by SVPD is blocked by an Spconfiguration, preventing cleavage of the upstream adjacentphosphorothioate linkage. With 2'-PDE, the A_(Rp) A_(Rp) A, A_(Sp)A_(Rp) A and A_(Rp) A_(Sp) A trimer cores, but not the A_(Sp) A_(Sp) Atrimer core, were substrates. With both SVPD and 2'-PDE, the dimer cores(either A_(Rp) A or A_(Sp) A) accumulate following hydrolysis of theA_(Rp) A_(Rp) A' A_(Sp) A_(Rp) A and A_(Rp) A_(Sp) A trimer cores.Hydrolysis of the 2-5A molecule by SVPD and 2'-PDE proceeds from the2'/3'-terminus. Therefore, introduction of the phosphorothioate groupinto the trimer core results in the accumulation of the A_(Rp) A orA_(Sp) A dimer cores from the 5'-terminus and the accumulation of AMPSfrom the 2'/3'-terminus (Table 3). With authentic A₃, there is nodetectable accumulation of A₂ following hydrolysis by SVPD or 2'-PDE.

None of the Sp linkage-containing trimer cores were cleaved by SVPD.While the half-life of A_(Sp) A_(Rp) A upon cleavage by L-cell extract(15 hours) did not differ significantly from that of A_(Rp) A_(Rp) A (18hours), the half-life for the A_(Rp) A_(Sp) A trimer was orders ofmagnitude longer (20 days). A_(Sp) A_(Sp) A was not cleaved by L-cellextract (Table 3).

Preparation of Phosphorothioate Tetramer Cores

The following non-limiting examples illustrate the preparation of thefully-resolved tetramer core compounds of the invention.

EXAMPLE 11

a.(Sp)-P-Thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-(Sp)-P-thiodenylyl-(2'-5)-adenosine

b.(Rp)-P-thiodenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-(Sp)-P-thiodenylyl-(2'-5')-adenosine.

A solution of 0.149 g (0.065 mmole) of fully protected trimer 8B, whichhas the stereoconfiguration SpSp, was detritylated with 2%p-toluenesulfonic acid in 1.5 ml dichloromethane/methanol (4:1) for 3 hat room temperature. The mixture was diluted with 50 ml CHCl₃, washedwith phosphate buffer (2×15 ml), dried (Na₂ SO₄), and evaporated todryness. The residue was chromatographed on silica gel plates (20×20×0.2cm) developed with dichloromethane/ethylacetate/n-hexane (5:5:3 v/v).The product band, R_(f) 0.55, was cut out, eluted withchloroform/ethanol (1:1) and gave, on evaporation to a colorless foam of0.108 g (yield 88%). The 5'-deblocked SpSp trimer 8B (101 mg; 0.05 mM)was dissolved in 0.5 ml acetonitrile overnight with phosphitamide 2(0.105 g; 0.1 mmole). 3-Nitro-1,2,4-triazole (0.023 g; 0.2 mmole) wasadded. After stirring at room temperature for 3 h, sulfur (0.042 g; 1.3mmole) in pyridine (0.084 ml) was added for oxidation. After stirring atroom temperature for another 24 h, the product was extracted withdichloromethane (50 ml), the organic phase was washed with saturatedNaCl solution (2×20 ml), dried over Na₂ SO₄, and then evaporated todryness. Final coevaporation was performed with toluene to removepyridine. The crude product was purified by using preparative silica gelplates (20×20×0.2 cm) to which about 50 mg per plate was applied foroptimal separation by developing indichloromethane/ethylacetate/n-hexane (1:1:0.5 v/v ) three times. Theband containing the fully protected SpSpSp tetramer (R_(f) 0.3) and theband containing the fully protected RpSpSp tetramer (R_(f) 0.4), werecut out and eluted with chloroform/methanol (4:1). The yield of thefully protected SpSpSp tetramer was 43 mg; 29%. The yield of the fullyprotected RpSpSp compound was 53 mg; 35.5%. The two isomers (7.3micromoles; 0.22 mg) were deblocked by stirring with 0.5M DBU inpyridine (5.0 ml) for 20 h, neutralized with 1M acetic acid/pyridine(0.5 ml) and finally evaporated. The subsequent desilylation wasachieved with 1M tetrabutylammonium fluoride in tetrahydrofuran (3.6 ml)for 48 h at r.t. The mixture was concentrated in vacuo, the residuedissolved in conc. ammonia (15 ml) and stirred at r.t. for 48 h. Afterevaporating the solution, the residue was taken up in 5 ml of 80% aceticacid and allowed to stand for 20 h at r.t. The residue was dissolved inabout 5 ml water and put on a DEAE Sephadex A-25 column (60×1 cm) andthe product was eluted with a linear gradient of Et₃ NH⁺ HCO₃ ⁻ buffer(pH 7.5) (gradient 0.001-1M). The product fractions were collected,evaporated and further purified by paper chromatography using;i-PrOH/conc. ammonia/water (6:1:3). The tetramer isomers were elutedwith water to give A_(Sp) A_(Sp) A_(Sp) A (72% yield; R_(f) 0.23) andA_(Rp) A_(Sp) A_(Sp) A (73% yield; R_(f) 0.29) as the ammonium salt.

EXAMPLE 12

a.(Rp)-P-Thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-adenosine

b.(Sp)-P-Thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-adenosine

The title compounds are prepared by following the procedure of Example11, but substituting the fully protected trimer 7A for 8B as thestarting material.

EXAMPLE 13

a.(Rp)-P-Thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-adenosine

b.(Sp)-P-Thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-(Rp)-P-thioadenylyl-(2'-5')-adenosine

The title compounds are prepared by following the procedure of Example11, but substituting the fully protected trimer 7B for 8B as thestarting material.

EXAMPLE 14

a.(Sp)-P-Thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-adenosine

b.(Sp)-P-Thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-(Sp)-P-thioadenylyl-(2'-5')-adenosine

The title compounds are prepared by following the procedure of Example11, but substituting the fully protected trimer 8A for 8B as thestarting material.

Preparation of 2',5'-Phosphorothioate Oligoadenylate 5'-Monophosphates

5'-Monophosphates of 2',5'-oligoadenylates are readily prepared byreacting the corresponding core compounds with POCl₃. Such treatmentwould result in the elimination of sulfur from the phosphorothioateinternucleotide linkages of the compounds of the present invention, andthe formation of 2-5A. Thus, the 5'-monophosphates of thephosphorothioate oligoadentylates must be prepared from thecorresponding fully protected core compounds from which themonomethoxytrityl blocking group on the 5'-terminal nucleotide has beenremoved. The conditions of the phosphorylation must be such that thep-nitrophenylethyl blocking groups on the internucleotide phosphorousatoms remain intact.

The 5'-monophosphate of each resolved trimer core of the presentinvention was prepared from the 5'-hydroxy analogue of the correspondingfully protected trimer 7A, 7B, 8A or 8B. The intermediate5'-phosphotriester (11A, 11B, 12A or 12B) was prepared according toExample 15 and then freed of all blocking groups according to Example 16to yield the 5'-monophosphate. The procedure of Examples 15 and 16 maybe used for forming the 5'-monophosphate of any of the four trimer corestereoisomers.

EXAMPLE 15

5'-O-(2,5-Dichlorophenyl-p-nitrophenylethyl)-phosphoryl-6-N-benzoyl-3'-O-tert-butyldimethylsilyl-P-thioadenylyl-2'-[O^(P)-(p-nitrophenylethyl)-5']-6-N-benzoyl-3'-O-tert-butyldimethylsilyl-P-thioadenylyl-2'-[O^(P)-(p-nitrophenylethyl)-5']-6-N-benzoyl-2',3'-di-O-tert-butyldimethylsilyladenosine (11A, 11B, 12A or 12B): To a solution of 1,2,4-triazole (0.011g; 0.16 mmole) and 2,5-dichlorophenylphosphorodichloridate (0.022 g;0.078 mmole) in dry pyridine (0.5 ml) was added the 5'-deblockedanalogue of either 7A, 7B, 8A or 8B (0.1 g; 0.049 mmole) (prepared as anintermediate in Example 11), and after stirring for 30 min,p-nitrophenylethanol (0.02g; 0.119 mmole) was added and stirringcontinued for 20 h. The solution was then extracted with chloroform (50ml), the organic phase was washed with water (2×20 ml), evaporated todryness, and finally co-evaporated with toluene. The residue waspurified by silica gel chromatography on preparative plates (20×20×0.2cm) using the system dichloromethane/n-hexane/ethylacetate (1:1:1 v/v).The product band was eluted with chloroform/methanol (4:1) andevaporated in vacuo to give 11A, 11B, 12A or 12B in 70-80% yield,respectively.

EXAMPLE 16

a.5'-O-Phosphoryl-(Rp)-thioadenylyl-(2'-5')-(Rp)-thioadenylyl-(2'-5')-adenosine(13A)

b.5'-O-Phosphoryl-(Sp)-thioadenylyl-(2'-5')-(Rp)-thioadenylyl-(2'-5')-adenosine(13B)

c.5'-O-Phosphoryl-(Rp)-thioadenylyl-(2'-5')-(Sp)-thioadenylyl-(2'-5')-adenosine(14A)

d.5'-O-Phosphoryl-(Sp)-thioadenylyl-(2'-5')-(Sp)-thioadenylyl-(2'-5')-adenosine(14B)

p-Nitrobenzaldoxime (0.036 g; 0.216 mmole) was stirred for 30 min indioxane/triethylamine/water (each 0.5 ml), the appropriate5'-phosphotriester 11A, 11B, 12A or 12B (0.05 g, 0.02 mmole) was addedand the mixture was kept at r.t. for 4 h. The solution was evaporated todryness, followed by coevaporation with toluene (2×5 ml), and theresidue purified by preparative TLC on plates (20×20×0.2 cm) inchloroform/methanol (95:5). The product band was eluted withchloroform/methanol/triethylamine (5:1:1) and evaporated to dryness.This material (0.022 g; 10 micromole) was stirred with 0.5M DBU inpyridine (8 ml) at r.t. for 24 h, the solution neutralized with 1Macetic acid (4 ml) and evaporated to dryness. The residue was treatedwith 1M Bu₄ NF in THF (6 ml) for 48 h and after evaporation thedebenzoylation was accomplished by treatment with conc. ammonia (25 ml)at r.t. for 48 h. The solution was evaporated. The deblocked crudetrimer 5'-monophosphate was taken up in water (25 ml) and washed withchloroform (2×10 ml). The aqueous phase put on a DEAE Sephadex A-25column (60×1 cm) for elution with a linear gradient of 0.001-1M Et₃ NH⁺HCO₃ ⁻ buffer. The product fractions were collected, evaporated todryness, and after several coevaporations with water were furtherpurified by paper chromatography using the i-PrOH/concammonia/water-system (55:10:35). The product band was eluted with waterand gave on lyophilization the trimeric P-thioadenylate 5'-monophosphate13A, 13B, 14A or 14B as ammonium salts in 68-74% yield. ##STR10##

The ¹ H-NMR of the 5'-monophosphates are as follows:

                                      TABLE 4                                     __________________________________________________________________________    .sup.1 H-NMR Spectra of 5'O-Phosphoryl-                                       P-thioadenylyl-(2'-5')-P-thioadenylyl-                                        (2'-5')-adenosine Stereoisomers.sup.2                                         Compound                                                                            1'-H        2-H      8-H      Solvent                                   __________________________________________________________________________    13A   6.04                                                                              5.92 d                                                                            5.75 d                                                                            8.31;                                                                            8.18;                                                                            8.12;                                                                            7.98;                                                                            7.85;                                                                            7.80                                                                             D.sub.2 O                                 13B   6.12 s                                                                            5.94 d                                                                            5.76 d                                                                            8.26;                                                                            8.21;                                                                            8.11;                                                                            7.99;                                                                            7.94;                                                                            7.86                                                                             D.sub.2 O                                 14A   6.03 s                                                                            5.92 d                                                                            5.80 d                                                                            8.27;                                                                            8.22;                                                                            8.15;                                                                            8.04;                                                                            7.93;                                                                            7.81                                                                             D.sub.2 O                                 14B   6.09 s                                                                            5.94 s                                                                            5.81 d                                                                            8.41;                                                                            8.26;                                                                            8.14;                                                                            8.07;                                                                            8.02;                                                                            7.89                                                                             D.sub.2 O                                 __________________________________________________________________________     .sup.2 δ values in ppm; Standard TMS; characteristic signals       

Monophosphorylation of the 5'-deblocked protected trimers to form the5'-phosphotriesters 11A, 11B, 12A or 12B proceeds in high yield, 70-80%,followed by the further high yield (68-74%) step of completedeprotection resulting in the trimer 5'-monophosphates 13A, 13B, 14A or14B.

The 5-monophosphates of each resolved tetramer core compound of thepresent invention is prepared in the same fashion, using the identicalmolar quantities as in Examples 15 and 16 except that the startingmaterial for the synthesis is the 5'-hydroxy analogue of the fullyprotected tetramer rather than the 5'-hydroxy analogue of the fullyprotected trimer. The following fully resolved tetramer5'-monophosphates are thus prepared:

5'-O-Phosphoryl-(Rp)-thioadenylyl-(2'-5')-(Rp)-thioadenylyl-(2'-5')-(Rp)-thioadenylyl-(2'-5')-adenosine

5'-O-Phosphoryl-(Rp)-thioadenylyl-(2'-5')-(Rp)-thioadenylyl-(2'-5')-(Sp)-thioadenylyl-(2'-5')-adenosine

5'-O-Phosphoryl-(Rp)-thioadenylyl-(2'-5')-(Sp)-thioadenylyl-(2'-5')-(Sp)-thioadenylyl-(2'-5')-adenosine

5'-O-Phosphoryl-(Sp)-thioadenylyl-(2'-5')-(Sp)-thioadenylyl-(2'-5')-(Sp)-thioadenylyl-(2'-5')-adenosine

5'-O-Phosphoryl-(Sp)-thioadenylyl-(2'-5')-(Rp)-thioadenylyl-(2'-5')-(Sp)-thioadenylyl-(2'-5')-adenosine

5'-O-Phosphoryl-(Sp)-thioadenylyl-(2'-5')-(Rp)-thioadenylyl-(2'-5')-(Rp)-thioadenylyl-(2'-5')-adenosine

5'-O-Phosphoryl-(Sp)-thioadenylyl-(2'-5')-(Sp)-thioadenylyl-(2'-5')-(Rp)-thioadenylyl-(2'-5')-adenosine

5'-O-Phosphoryl-(Rp)-thioadenylyl-(2'-5')-(Sp)-thioadenylyl-(2'-5')-(Rp)-thioadenylyl-(2'-5')-adenosine

Preparation of 2',5'-Phosphorothioate Oligoadenylate 5'-Diphosphates and5'-Triphosphates

The 5'-diphosphate and 5-triphosphate of the 2',5'-phosphorothioateoligoadenylates may be prepared from the 5'-monophosphate by followingthe procedure of Example 17.

EXAMPLE 17

All reactions are performed in glassware oven-dried at 125° C. for 18-24hr. A 2',5'-phosphorothioate oligoadenylate stereoisomer (trimer ortetramer, 400 OD units at 260 nm) is dissolved in 500 microliters of drydimethylformamide ("DMF") and dried in vacuo in a 10 ml conical flask at35° C. This process is repeated three times. To the dry residue, 50micromoles of triphenylphosphine, 100 micromoles of imidazole and 50micromoles of dipyridinyl disulfide are added. The mixture is dissolvedin 500 microliters dry DMF plus 50 microliters of dry dimethylsulfoxide.The solution is stirred with a stirring bar for 2 hr at roomtemperature. After 2 hr the solution is homogeneous (after 30 minutes,the solution begins to change to yellow). The solution is transferreddropwise to 10 ml of a 1% NaI/dry acetone (w/v) solution. The clearwhite precipitate which forms is the sodium salt of the5'-phosphoroimidazolidate. The precipitate is centrifuged at roomtemperature, the supernatant is decanted, and the precipitate is washedthree times with 10 ml dry acetone. The centrifuging is repeated. Theprecitipate is dried over P₂ O₅ in vacuo for 2 hr. The precipitate isdissolved in 200 microliters of freshly prepared 0.5M tributylammoniumpyrophosphate in dry DMF. The solution is maintained at room temperaturefor 18 hr after which time the DMF is removed in vacuo. The residue isdissolved in 0.25M triethylammonium bicarbonate buffer ("TEAB") (pH7.5). The 5'-di and 5'-triphosphate products are separated using aDEAE-Sephadex A25 column (HCO₃ ⁻ form; 1×20 cm) with a linear gradientof 0.25M to 0.75M TEAB. Fractions (10 ml) are collected. The product isobserved by ultraviolet spectroscopy at 254 nm. The fractions containingthe 5'-di- and 5'-triphosphates are separately pooled and dried invacuo. The TEAB is removed by repeated addition of water followed bylyophilization. The yield of the 5'-diphosphate is about 5%; the yieldof the 5'-triphosphate is about 60%.

It is generally regarded that activation of RNase L by 2-5A is key tothe antiviral defense mechanisms. Interferon induces transcription ofthe enzyme 2-5A synthetase which produces 2',5' linked oligoadenylatesupon activation of double-stranded RNA. The only known biochemicaleffect of 2-5A is activation of RNase L. This enzyme hydrolyses mRNA andrRNA, thereby resulting in inhibition of protein synthesis. Theactivation of RNase L is transient unless 2-5A is continuouslysynthesized, since 2-5A is rapidly degraded. RNase L activation thusplays a critical role in inhibiting replication, and therefore indefending against infection by viruses.

According to the invention, all four of the 2',5'-phosphorothioateadenylate trimer cores, and their 5'-monophosphates bind to RNase L, asdetermined by radio binding assay according to the method of Knight etal, Meth. Enzymol. 79:216-227 (1981). The 2',5'-phosphorothioateadenylate trimer cores and authentic A₃ were able to displace p₃ A₄ [³²P]pCp probe from RNase L in L929 cell extracts in aconcentration-dependent manner (FIG. 1A). IC₅₀ s varied from 2×10⁻⁶ to5×10⁻⁶ M. However, the 5'-monophosphorylated trimers had 1000-foldhigher binding affinity to RNase L than their respective cores, that is,IC₅₀ s ranged from 2×10⁻⁹ to 5×10⁻⁹ M (FIG. 1A). Without wishing to bebound by any theory, this increase may be attributed to the ability ofthe 5'-monophosphates to anchor the molecule to RNase L more effectivelybecause of increased polarity.

The 2',5'-phosphorothioate cores, with one exception, have the correctstereoconfiguration to activate RNase L. The activation ofpartially-purified PNase L by the 2',5'-phosphorothioates was measuredaccording to the core-cellulose assay of Silverman, Analyt. Biochem.144:450-460 (1985) which relies on hydrolysis of the substratepoly(U)-3-[³² P]pCp. Surprisingly, three of the four2',5'-phosphorothioate adenylate cores were able to activate RNase L tocleave poly(U)-3'-[³² P]pCp in the core-cellulose assay. (FIG. 1B) Theorder of activation of the trimer cores and the corresponding5'-monophosphates is: RpRp>SpRp>RpSp (FIG. 1B). While pA_(Rp) A_(Rp) Awas the most efficient activator of RNase L, the compound ismetabolically unstable and is readily attacked by phosphodiesterases(See Table 3). The SpSp trimer core did not activate RNase L, even at aconcentration of 10⁻³ M. As was observed in the binding assay (FIG. 1A),there was a 1000-fold increase in the activation of RNase L by the5'-monophosphates of the 2' ,5'-phosphorothioate trimers compared totheir respective cores.

Activation of RNase L by 2',5'-phosphorothioate adenylate trimer coresand their 5'-monophosphates was also measured in an rRNA cleavage assayusing L929 cell extracts. A_(Rp) A_(Rp) A and A_(Sp) A_(Rp) A activatedRNase L to cleave 28S and 18S rRNA to specific cleavage products at 10⁻⁵M. However, A_(Rp) A_(Sp) A and A_(Sp) A_(Sp) A did not activate RNase Lat concentrations as high as 10⁻⁴ M. It appears that the rRNA cleavageassay was not sensitive enough to detect activation of RNase L by A_(Rp)A_(Sp) A. Under the experimental conditions used, authentic A₃ core wasalso inactive, which is in agreement with previous reports (Haugh et al,Eur. J. Biochem. 132:77-84 (1983)).

The corresponding 5'-monophosphates pA_(Rp) A_(Rp) A, pA_(Sp) A_(Rp) A(at 10⁻⁸ M) and pA_(Rp) A_(Sp) A (at 10⁻⁷ M) activated RNase L to cleave28S and 18S rRNA. Authentic pA₃ was active at 10⁻⁶ M. Incubation withpA_(Sp) A_(Sp) A, even at concentrations as high at 10⁻⁵ M, did notresult in detectable rRNA degradation.

The increased bonding strength of the 5'-phosphorylated trimer coreA_(Rp) A_(Sp) A provides a relatively metabolically stable and highlyefficient activator for RNase L.

A_(Sp) A_(Sp) A and corresponding 5'-monophosphate were observed toinhibit RNase L activation in both the core-cellulose and rRNA cleavageassays. Notwithstanding, these compounds are extremely useful as probesin the evaluation of the role of RNase L in the interferon-inducedbiological cascade. Most importantly, pA_(Sp) A_(Sp) A selectivelyinhibits activation of RNase L at physiological concentrations, and ismetabolically stable to specific and non-specific phosphodiesterases.The molecule provides the means to selectively shut off RNase Lactivation.

pA_(Sp) A_(Sp) A is the most effective inhibitor of RNase L reported todate. Moreover, notwithstanding its RNase L inhibitory effect, pA_(Sp)A_(Sp) A is observed to inhibit HIV reverse transcriptase activity andtobacco mosaic virus replication.

For pharmaceutical use, the compounds of the invention may be taken upin pharmaceutically acceptable carriers, such as, solutions,suspensions, tablets, capsules, ointments, elixirs and injectablecomposition and the like. They are administered to subjects sufferingfrom viral infection. The dosage administered depends upon the natureand severity of the infection, the disease stage, and, when administeredsystematically, the size and weight of the infected subject.

The compounds are generally administered in the form of water-solublesalts. Pharmaceutically acceptable water soluble salts include, forexample, the sodium, potassium or ammonium salts of the activecompounds. They are readily dissolved in water or saline solution. Thus,the preferred formulation for pharmacological use comprises a salinesolution of the desired compound in salt form. The formulation mayfurther contain an agent, such as a sugar or protein, to maintainosmotic balance. The salt form of the compound is preferred owing to therelatively high acidity (about pH 3) of the acid form of the compounds.

The compounds may be applied topically. A sufficient amount of apreparation containing a compound of the invention is applied to coverthe lesion or affected area. An effective concentration of active agentis between about 10⁻³ M and 10⁻⁵ M, with 10⁻⁴ M being preferred.

Effect of 2',5'-Phosphorothioate Oligoadenylates on HIV ReverseTranscriptase Activity

The compounds of the invention inhibit the reverse transcriptaseactivity of HIV.

HIV reverse transcriptase (RNA-dependent DNA nucleotidyl-transferase)activity was assayed by a modification of the procedure of Poiesz et al.(Poiesz, B. J., Ruscetti, F. W., Gazdar, A. F., Bunn, P. S., Minna, J.D., and Gallo, R. C., Proc. Natl. Acad. Sci. U.S.A. 77:, 7415-7419(1980)).

Cultured H-9 cells are grown at 10⁶ cells/ml in RPMI-1640 medium and 20%heat-inactivated fetal calf serum. Cell suspensions are centrifuged(1000×g, 10 min.) and the supernatant is removed. Virus particles areprecipitated from this cell-free supernatant to which 0.3 ml of 4M NaCland 3.6 ml of 30% (weight/volume) polyethylene glycol are added. Thesuspension is placed on ice for 2 hr following centrifugation at15,000×g for 30 min at 0° C. The precipitate is resuspended in 200microliters of 50% glycerol (vol./vol.)/25 mM Tris-HCl (pH 7.5)/5 mMdithiothreitol/50 mM KCl/0.025% Triton X-100. Virus particles are lysedby the addition of 100 microliters of 0.9% Triton X-100/1.5M KCl.Reverse transcriptase assays are performed at 37° C. for 1 hr with 10microliters of the lysed virus solution in a final reaction volume of100 microliters containing: 40 mM Tris-HCl (pH 7.8), 4 mMdithiothreitol, 45 mM KCl and 2.5 micrograms of template primer[poly(A)-dT₁₅, 0.5 micrograms/microliter] (with a final Mg⁺⁺concentration of 10 mM). At this time, 10 microliters of the2',5'-phosphorothioate oligoadenylate is added to a final concentrationof 200 micromolar. Reaction mixtures also contain 4 micromoles of [³H]dTTP. Reactions are stopped by the addition of cold 5% trichloroaceticacid and filtered through nitrocellulose discs. The discs are dried andthe radioactivity bound to the discs is determined. Reversetranscriptase activity is expressed as the percent relative to acontrol.

The data is shown in Table 5. The compounds were assayed in the abovemanner in two sets, each set having a separate set of controls. Thecompounds of reactions 1-4 were assayed against control #1, which was187×10³ cpm. The compounds used in reactions 5-13 were assayed againstcontrol #2, which was 273×10³ cpm.

                  TABLE 5                                                         ______________________________________                                        Inhibition of HIV (HTLV-III.sub.BH-9) Reverse                                 Transcriptase Activity by 2',5'-                                              Phosphorothioate Oligoadenylates                                                                          Reverse Trans-                                                      Concentra-                                                                              criptase  Percent                                 Reaction          tion      Activity  Inhibi-                                 No.    Compound   (μM)   (cpm × 10.sup.-3)                                                                 tion                                    ______________________________________                                        1      p.sub.3 A.sub.3                                                                          200       187       0                                       2      pA.sub.3   200       133       29                                      3      A.sub.3    200       201       0                                       4      pA.sub.Rp A.sub.Rp A                                                                     200       144       25                                      5      pA.sub.Sp A.sub.Rp A                                                                     200       230       16                                      6      pA.sub.Rp A.sub.Sp A                                                                     200       206       25                                      7      pA.sub.Sp A.sub.Sp A                                                                     200       232       15                                      8      A.sub.Rp A.sub.Rp A                                                                      200       224       18                                      9      A.sub.Sp A.sub.Rp A                                                                      200       258       6                                       10     A.sub.Rp A.sub.Sp A                                                                      200       247       10                                      11     A.sub.Sp A.sub.Sp A                                                                      200       219       20                                      12     A.sub.Sp A.sub.Sp A.sub.Sp A                                                             200       149       45                                      13     A.sub.Rp A.sub.Sp A.sub.Sp A                                                             200       147       46                                      ______________________________________                                    

Although p₃ A₃, pA₃ and A₃ are found in mammalian cells, only themonophosphate inhibits HIV reverse transcriptase. The core compounds ofthe present invention on the other hand are observed to inhibit HIVreverse transcriptase. While all the compounds of the invention(reaction nos. 5,6,7,9,10,11,12 and 13) inhibit the transcriptase tosome degree, the tetramers are particularly effective.

The compounds of the invention may be administered in amounts of fromabout 10 micromoles to about 200 micromoles.

The compounds also possess antiviral activity against plant-infectingvirus, particularly tobacco mosaic virus. Similar results may beobtained against other viruses which cause necrosis in turnips,cucumber, orchids and in other plants. Such viruses include, but are notlimited to, tobacco vein mottling virus, vesicular stomatitis virus,vaccinia virus, turnip necrosis virus, and cymbidium orchid virus.

The compounds may be administered effectively to plants by topicalapplication by abrasion of the leaf surface, aerosol spray, treatment ofthe soil, spraying, or dusting.

An effective antiviral composition may be formed by combining one ormore of the compounds of the invention with a suitable carrier material.While the individual stereoisomers are preferred for pharmaceutical use,mixtures of one or more of stereoisomers may be employed in agriculturalapplications. The active compound may also be administered by sprayinginsect vectors such as aphids, thrips and whiteflies which carry virusto plants. The dosage administered depends upon the severity of theinfection.

The compounds of the invention may be applied to plant seeds prior togermination to control viruses contained in the germ plasm. The seedsmay be soaked in a solution of polyethylene glycol ("PEG") containingone or more of the compounds. PEG brings the seeds to physiologicalactivity and arrest. The relative concentration of active compound toPEG depends upon the type of seed under treatment.

Plants are effectively treated with an aqueous formulation containingfrom about 10⁻¹ to about 10⁻² M concentration of active ingredient. Thecompounds of the invention may be applied at very low concentrations. Aneffective amount of active ingredient on the plant surface is from about10⁻⁸ to about 10⁻¹² mole per cm² of plant surface area, with about 10⁻¹⁰mole to about 10⁻¹² mole per cm² being preferred. For the typicaltobacco plant of 1,000 cm², 10⁻⁵ M of compound is effective. At thisrate, one pound of active ingredient is sufficient to treat 2×10⁸tobacco plants.

For agricultural application, the compounds are advantageouslyadministered in the form of water-soluble salts, e.g. ammonium orpotassium salts. Sodium salts are generally avoided in treating edibleplants.

The compounds of the invention are readily dissolved in water,particularly at such low concentrations. Aqueous formulations foragricultural use may optionally contain a sticker and/or aUV-stabilizer. Such agents are well-known to those skilled in the art.Fatty acids (1%) are useful as spreader sticker agents. EffectiveUV-stabilizers include, for example, p-aminobenzoic acid.

Effect of 2',5'Phosphorothioate Oligoadenylates on Tobacco Mosaic Virus(TMV) Replication in Intact Tobacco Plants

The effectiveness of the 2',5'-phosphorothioate oligoadenylates againstplant virus was demonstrated by infectivity tests on intact Nicotianaglutinosa plants as follows.

Carborundum (400 mesh ) was sprinkled lightly onto leaves. Solutionscontaining 0.2 micrograms per ml of TMV and 2×10⁻⁵ M2',5'-phosphorothioate oligoadenylate in phosphate buffer were appliedonto half-leaves of N. glutinosa with either a gloved finger or with apipettor. The remaining half-leaves were controls (inoculated with thebuffer solution containing TMV, but no active compound). The infectionwas allowed to proceed 48 h under continuous illumination of about 1500Lx at which time local virus lesions appeared. Inhibition of TMVreplication was calculated as the percent of local lesions produced in2',5'-phosphorothioate oligoadenylate-treated half-leaves compared tocontrol half-leaves. The data is set forth in Table 6.

                  TABLE 6                                                         ______________________________________                                        Compound.sup.3                                                                             Percent TMV Inhibition.sup.4                                     ______________________________________                                        --           0                                                                A.sub.3      16                                                               A.sub.Rp A.sub.Rp A                                                                        57                                                               A.sub.Sp A.sub.Sp A                                                                        80                                                               A.sub.Rp A.sub.Sp A                                                                        70                                                               A.sub.Sp A.sub.Rp A                                                                        33                                                               pA.sub.Rp A.sub.Rp A                                                                       75                                                               pA.sub.Rp A.sub.Sp A                                                                       15                                                               pA.sub.Sp A.sub.Rp A                                                                       23                                                               pA.sub.Sp A.sub.Sp A                                                                       40                                                               A.sub.Sp A.sub.Sp A.sub.Sp A                                                               70                                                               A.sub.Rp A.sub.Sp A.sub.Sp A                                                               80                                                               ______________________________________                                         .sup.3 All compounds were added at 2 × 10.sup.-5 M in the infecting     solution.                                                                     .sup.4 Inhibition of TMV replication was detected 48 hr after infection       and was calculated as the percent of local lesions produced in treated        halfleaves of N. glutinosa compared to control halfleaves.               

Non-infected plants were treated with 2×10⁻⁶ M and 2×10⁻⁵ M of the2',5'-phosphorothioate core and 5'-monophosphate analogues. No toxicity(chlorosis or necrosis) was observed during the two week period tested.

Of the ten phosphorothioate trimer and tetramer cores and5'-monophosphates tested, the SpSp phosphorothioate trimer coreinhibited TMV replication to the greatest extent (80%). Similarly, thecorresponding tetramer core, SpSpSp, inhibited TMV replication by 70%.The RpSpSp tetramer core inhibited TMV replication by 80%. Theremarkable inhibition by A_(Sp) A_(Sp) A, A_(Sp) A_(Sp) A_(Sp) A andA_(Rp) A_(Sp) A_(Sp) A, as well as the 70% inhibition by A_(Rp) A_(Sp)A, compares with only a 16% inhibition with authentic A₃. By introducingthe property of chirality, as well as increased metabolic stability,there is a marked increase of the inhibition of TMV replication by thecompounds of the invention over 2-5A.

In addition to administration with conventional carriers, the compoundsof the present invention may be administered by a variety of specializedoligonucleotide or nucleic acid delivery techniques. 2-5A and itsanalogues have been successfully encapsulated in unilamellar liposomesand delivered with the aid of monoclonal antibodies to cells (Bayard etal, Eur. J. Biochem. 151:319-325 (1985)). Reconstituted Sendai virusenvelopes have been successfully used to deliver RNA and DNA to cells(Arad et al, Biochem. Biophys. Acta. 859:88-94 (1986)). These techniquesmay be utilized for introduction of the present 2',5'-phosphorothioateoligoadenylates into cells.

It is further contemplated that the compounds of the invention may beadministered in the form of prodrugs in which lipophilic groups areattached to, for example, the 5'-terminal hydroxyl group of the corecompound.

Conjugation of 2',5'-Phosphorothioate Tetramer Adenylates

Poly(L-lysine) has been described as a versatile membrane carrier for2-5A and other macromolecules (Bayard et al, Biochem. 25:3730-3726(1986)). The tetramer cores and phosphorylated tetramers of the presentinvention may be conveniently administered in the form of poly(L-lysine)conjugates. The conjugates are formed by introducing two aldehydefunctions at the 2' end of the tetramer by periodate oxidation of thealpha-glycol group of the ribose residue. The resulting aldehyde groupsare then randomly coupled to the epsilon-amino groups of lysine residuesof poly(L-lysine) by Schiff base formation, and then reduced with sodiumcyanoborohydride at pH 8.0. This procedure converts the 2',3'-terminalribose ring into a morpholine structure. The poly(L-lysine) peptidepreferably contains from about 60 to about 70 lysine residues. Fromabout five to about ten of the lysine residues are coupled in thismanner to tetramer moieties. The resulting2',5'-phosphorothioate/poly(L-lysine) conjugates may then be isolated bygel filtration chromatography on a Sephadex G-50 column.

The poly(L-lysine)/2',5'-phosphorothioate oligoadenylate conjugates havethe formula: ##STR11## wherein q is an integer from about 60 to about 70and R is randomly R' or ##STR12## From about five to about ten of the Rgroups comprise R'. The R' group has the following formula wherein m is0,1,2 or 3: ##STR13##

The conjugates may be advantageously prepared by the procedure of Bayardet al, Biochem. 25:3730-3736 (1986):

EXAMPLE 18 Preparation of Poly(L-lysine)/2',5'-PhosphorothioateOligoadenylate Conjugates

A 4-microliter aliquot of sodium metaperiodate (0.6 micromole in 0.1Msodium acetate buffer, pH 4.75) is added to an ice-cold solution of2',5'-phosphorothioate tetramer adenylate in 400 microliter of distillerwater. The reaction mixture is stirred on ice for 30 min; 400 microliterof poly(L-lysine) (0.14 micromole in 0.2M phosphate buffer, pH 8.0) and200 microliter of sodium cyanoborohydride (20 micromole in 0.2Mphosphate buffer, pH 8.0) are added. The mixture is incubated for 2 h atroom temperature and then loaded on a Sephadex G-50 column equilibratedwith 0.1M sodium acetate buffer, pH 4.75. Each fraction is assayed forits phosphorothioate oligoadenylate/poly(L-lysine) content by the methoddescribed by Lowry et al, J. Biol. Chem. 193:265-275 (1951), and byabsorbance at 260 nm.

Conjugation of the 2',5'-phosphothioate tetramer to poly(L-lysine)leaves the remaining three 2',5'-linked phosphorothioate adenylicresidues intact for optimal RNase L binding and activation.

Liposome Encapsulation of 2',5'-Phosphorothioate Oligoadenylates

Encapsulation of the compounds of the present invention comprisesanother attractive non-disruptive technique for introduction into cells.Liposome encapsulation may be advantageously accomplished according tothe technique described by Kondorosi et al., FEBS Lett. 120:37-40(1980):

EXAMPLE 19 Preparation of Large Unilamellar Vesicles (Liposomes) Loadedwith 2',5'-Phosphorothioate Oligoadenylates

Briefly, a phospholipid mixture from bovine brain (Sigma Chemical Co.,Folch fraction III composed of 80-85% phosphatidylserine with theremaining 15% composed of other brain lipids; 35 mg) is suspended in 5ml of buffer A [0.1M NaCl, 2 mM histidine, 2 mMN-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid ("TES"), 0.4 mMEDTA (pH 7.4) by vortexing. The suspension is sonicated under nitrogenfor 10 minutes at 0° C. The suspension is further incubated for 1 hr at37° C. after adjusting the final concentration of Ca⁺⁺ to 20 mM by theaddition of 125 microliters of 800 mM CaCl₂. The resulting precipitateis sedimented by centrifugation (2500×g, 10 min), vortexing and mixingwith 100 microliters of 1×10⁻⁴ M 2',5'-phosphorothioate oligoadenylate,which is dissolved in phosphate-buffered saline. The final concentrationof EDTA is then adjusted to 120 mM by the addition of 400 microliters ofbuffer B [150 mM EDTA, pH 7.4, 0.1M NaCl, 2 mM histidine, 2 mM TES].Liposomes are formed after incubation of this mixture for 30 minutes at37° C. The excess of EDTA and non-encapsulated components are removed bypassing the liposomes through a Sephadex G-25 column which isequilibrated with phosphate-buffered saline. About 10% of the2',5'-phosphorothioate oligoadenylate is encapsulated into liposomes bythis procedure. The liposome suspension is stable at 4° C. for one weekfollowing preparation.

Preparation of Reconstituted Sendai Virus Envelopes Containing2',5'-Phosphorothioate Oligoadenylates

Reconstituted Sendai virus envelopes may be used as efficient vehiclesfor the introduction of polynucleotides into cells. Arad et al,Biochimica et Biophysica Acta 859:88-94 (1986) disclose introduction ofpoly(I) . poly(C) into cultured cells by the use of reconstituted Sendaivirus envelopes. Fusion of thus-loaded reconstituted Sendai virusenvelopes leads to introduction of the enclosed macromolecules into therecipient cell cytoplasm.

Reconstituted Sendai virus envelopes may be obtained by detergentsolubilization of intact Sendai virus particles. The reconstitutedenvelopes are fusogenic vesicles consisting of the viral envelopephospholids and their glycoproteins, devoid of the viral genomic RNA.

Incorporation of the compounds of the present invention intoreconstituted Sendai virus envelopes for fusion-mediated micro-injectionmay be accomplished by following the procedure or Arad et al. Briefly, apellet of Sendai virus particles (1.5 mg protein) is dissolved in 30microliters of a solution containing 10% Triton X-100, 100 mM NaCl, 50mM Tris-HCl (pH 7.4) and 0.1 mM phenylmethylsulfonyl fluoride (TritonX-100:protein ratio, 2:1, w/w ). To the clear supernatant obtained aftercentrifugation, 2',5'-phosphorothioate oligoadenylate dissolved in asolution A (160 mM NaCl, 20 mM Tris-HCl, (pH 7.4) ) is added to give afinal concentration of active ingredient of 5-20 mg/ml and a finalvolume of 150 microliters. Triton X-100 is removed from the supernatantby direct addition of 40 mg of SM-2 Bio-Beads. The turbid suspensionobtained (containing reconstituted Sendai virus envelopes) iscentrifuged at 100,000×g for 1 h. The pellet, containing about 10% ofthe original viral protein, is then suspended in solution A to give afinal protein concentration of 25 micrograms/ml.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indicating the scope of theinvention.

We claim:
 1. A mixture of two or more optical isomers of a compound ofthe formula ##STR14## wherein m is zero, 1, 2 or 3 and n is 2, andwater-soluble salts thereof.
 2. A conjugate of poly(L-lysine) and a2'-5'-phosphorothioate oligoadenylate, said conjugate having the formula##STR15## wherein q is an integer from about 60 to about 70 providedeach R is independently R' or ##STR16## and from about five to about tenof the R groups are R', which R' has the following formula wherein m is0,1,2 or 3: ##STR17##
 3. A conjugate according to claim 2 wherein the2',5'-phosphorothioate oligoadenylate from which said conjugate isformed is an optical isomer of the formula ##STR18## substantially freeof contamination of other optical isomers of the same formula, where mis zero, 1, 2, or 3, and at least one of the internucleotidephosphorothioate groups ##STR19## is of the SP configuration; orwater-soluble salt thereof.